APOPTOZA I METODY JEJ IDENTYFIKACJI PDF

The role of the process of apoptosis is investigated in the pathogenesis of many autoimmune diseases; however at present, there is not much information about its role in dermatitis herpetiformis. Skin biopsies were taken from 18 DH patients and from 10 healthy subjects. Expression of Bax, Fas, and Fas ligand was detected in the keratinocytes in skin biopsies from DH patients. The expression of examined molecules in biopsies from healthy people was observed only in single cells.

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The role of the process of apoptosis is investigated in the pathogenesis of many autoimmune diseases; however at present, there is not much information about its role in dermatitis herpetiformis. Skin biopsies were taken from 18 DH patients and from 10 healthy subjects.

Expression of Bax, Fas, and Fas ligand was detected in the keratinocytes in skin biopsies from DH patients. The expression of examined molecules in biopsies from healthy people was observed only in single cells. There were statistically significant differences between lesional, perilesional, and healthy skin of control group in Bax expression analysis and between lesional skin and control group in Fas, FasL, and TRAIL expression. There were statistically significant differences between control group and perilesional skin in Bax and FasL expression.

Our results show that selected proapoptotic molecules may take part in pathogenesis of dermatitis herpetiformis, but the role of apoptosis in this process is not clear. Dermatitis herpetiformis DH is a chronic subepidermal autoimmune bullous diseases characterized by skin and intestinal lesions.

Skin lesions include polymorphic eruption papules, vesicles , mainly distributed over the shoulders, elbows, backs, buttocks, and knees. They are usually symmetric and accompanied by severe pruritus. These symptoms are usually associated with asymptomatic and gluten-sensitive enteropathy.

Skin lesions in DH are histologically characterized by neutrophilic infiltrate leading to destruction of basement membrane zone BMZ proteins.

Impairment of type IV collagen, laminin, and entactin results in degradation of anchoring fibres and blister formation. The role of the process of apoptosis is investigated in the pathogenesis of many autoimmune diseases; however at present, there is not much information about its role in subepidermal blistering diseases.

It prevents many pathological processes, for example, autoimmunization and neoplasm [ 3 , 4 ]. Up to this day, two pathways of apoptosis have been described, that is, intrinsic and extrinsic and the basis for its distinction is a way of activation of the procaspases which initiate them [ 5 , 6 ]. The extrinsic pathway is associated with attaching the ligands to the receptor belonging to the superfamily of TNF receptors, which possess the so-called death domain DD , by means of which the activation of the procaspases inside the cell occurs [ 3 ].

The intrinsic and extrinsic pathways stimulating apoptosis are linked to each other by, for example, Bid protein, belonging to Bcl-2 family [ 6 ]. The proteins composing the superfamily of TNF receptor also take an active part in the process of apoptosis. The pathway of operation of the Fas ligand on the receptor Fas has been most thoroughly described. At present, it is believed that the constitutive coexpression of the receptor and the ligand Fas takes place in the cells of the rapid apoptotic turnover [ 9 ].

As TNF-R, Fas, and TRAIL receptors appear on the keratinocytes, they can take part in the pathogenesis of some skin diseases such as: toxic epidermal necrolysis, Graft-versus-host disease, skin neoplasms, and contact oversensitivity [ 8 ]. In the tissue material ex vivo , the apoptosis is difficult to determine quantitatively because of the dynamics of the process; therefore, the number of the registered apoptotic cells often constitute only a small percentage of the total number of cells which entered the state of apoptosis [ 10 ].

In general, the majority of cells of the hematopoietic line atrophies in the process of apoptosis and manifests the typical features of apoptosis, while the death of the epithelial cells is more complicated and very often hard to classify [ 11 ]. The patients were before treatment, at an active stage of the disease, that is, with skin lesions erythemas, papules, vesicles developed.

The lesions were accompanied by itch of different intensification. DH was diagnosed based on clinical picture, histological, and immunological findings. All the participants of the experiment gave an explicit consent in writing before entering the study and the study protocol was approved by The Local Ethical Committee of Medical University of Lodz no. The biopsies from all patients were taken from lesional and from uninvolved skin trunk before administration of any treatment topical or systemic.

In control group, biopsy specimens were taken from buttock or abdominal skin of healthy volunteers. Activity of endogenous peroxidase was inhibited with 0. Primary antibody solution directed against human antigens was put on these sections. DAKO EnVision double-step visualization system was then used in order to visualize the antigen-antibody reaction.

In cases of positive immunohistochemical reaction, cellular nuclei were stained with Meyer haematoxylin for 2 minutes. After dehydratation and processing through series of acetones and xylenes, the sections were fixed in DPX.

For every antibody a negative control was performed using TRIS buffer instead of antibody. In each specimen staining intensity of TRAIL and TRAIL receptor, Fas as well as Fas ligand in the epithelium and inflammatory infiltrates were recorded by two independent observers in 4—7 adjacent high power fields and graded from 0 staining not detectable , 1 minimal immunostaining in some cells , 2 weak immunostaining intensity in most cells , and 3 strong staining in most cells.

The mean grade was calculated by averaging grades assigned by the two authors and approximating the arithmetical mean to the nearest unity. Morphometric analysis was used for Bax MultiScan 8. The percentage of Bax immunopositive cells was estimated by counting in each slide cells in 4—7 adjacent high power fields semiautomatic function. Student's t -test was applied where appropriate after evaluation of distribution.

Mann-Whitney test was used where necessary. Bax protein expression was discovered in the cytoplasm of keratinocytes in samples of lesional skin mean immunoexpression The expression was weaker in uninvolved skin Fas expression in lesional skin was detected in the cytoplasm of keratinocytes 0.

In uninvolved skin, the expression was less intense 0. None of the samples taken from healthy patients revealed Fas expression. Fas ligand expression was revealed in the basal layer of the epidermis and inflammatory infiltrates in lesional skin 0.

In uninvolved skin, the expression was weaker in the basal layer of the epidermis and in few cells infiltrating the skin 0. Immunostaining for Fas ligand was negative in control group. Immunostaining of TRAIL was detected in the cytoplasm of keratinocytes as well as in inflammatory cells, and some fibroblasts in lesional skin 1. In uninvolved skin, the expression was detected in keratinocyte, mainly of the basal layer, and in some fibroblasts was less intense 0. As for healthy skin, the expression was discovered in keratinocytes and in some fibroblasts 0.

TRAIL immunoexpression in the cytoplasm of keratinocytes and inflammatory cells, score 1 , x. Immunostaining of TRAIL receptor was revealed in inflammatory infiltration, in the cytoplasm of keratinocytes as well as in some fibroblasts in lesional skin 0.

There were statistically significant differences between lesional, perilesional, and healthy skin of control group in Bax expression analysis. FasL expression was significantly higher in skin lesions and perilesional skin than in control group.

Statistical analysis was presented in Table 1. Expression of proteins in examined tissues. Statistical results. Apoptosis is claimed to be involved in a number of chronic inflammatory and neoplastic skin diseases such as contact dermatitis, toxic epidermal necrolysis, acantholytic dermatoses, and systemic lupus erythematosus [ 3 , 4 , 12 , 13 ], also may play role in pathogenesis of bullous diseases, although there are only a few works considering apoptosis in DH.

Mikalowski [ 14 ] showed cytolysis and desmosomal alterations in basal keratinocytes in DH skin lesions, that is a sign of cellular damage. Caproni et al. Apoptosis was seen in the basal and suprabasal layers of epidermis. In dermis, in the area of blood vessels, there were only a few apoptotic cells. In skin biopsies taken from the healthy control group there were no apoptotic cells. Proapoptotic Bcl-2 family consists of many proteins for example, Bax, that takes part in intracellular pathway of apoptosis.

In nonapoptotic cells, Bax and Bcl-2 form heterodimers keeping the homeostasis [ 6 ]. In our study, Bax expression was detected in keratinocytes of suprabasal layers, which was observed in earlier studies [ 16 ]. In our study similar to Caproni et al. Bcl-2 expression was present in epidermis and in dermis near superficial vessels.

The results of our study showed significant difference of Bax expression between lesional, perilesional, and healthy skin; that is why the overexpression of Bax protein seems to take part in DH pathogenesis, but explanation of the precise mechanism of starting the intrinsic pathway of apoptosis needs more studies.

In physiological condition, Fas expression is weak and is present in cell membrane, or in intracellular compartment, which prevents spontaneous apoptosis [ 17 — 19 ]. Although in physiological condition FasL is present on keratinocytes in granular layer, spinous layer and external hair shaft however, neither we, nor Caproni et al.

Also in other bullous diseases, Fas was examined, Morawej et al. Also our earlier research [ 21 ], considering pemphigoid, showed overexpression of Fas and FasL in lesional skin.

Studies of other authors showed FasL on activated T cells, neutrophils, natural killer cells, and macrophage lineage other cell types. The connection of ligand to Fas receptor triggers intracellular cascade leading to apoptosis.

Expression of both Fas on keratinocytes and FasL cells in the same topographic place implies that after the ligand and receptor have joined apoptosis gets started. Similar data showed Caproni et al. Higher expression of epidermal Fas than FasL and inversely in dermis, in the authors' opinion, indicates importance of extrinsic pathway of apoptosis in skin lesion formations [ 15 ].

Fas has not only proapoptotic activity, but also proinflammatory one [ 24 , 25 ]. Inflammatory activity of Fas showed Farley et al. This way of activation cannot be excluded in DH, because overexpression of many proinflammatory cytokines e.

Many factors, such as UV radiation, cytokines e. More precise studies are needed to establish which of these factors can take part in DH.

Although both of them coexist on keratinocytes, DR4 seems to be more effective [ 32 ]. These receptors do not take part in terminal differentiation of keratinocytes [ 33 ]. That leads to lymphocyte adhesion [ 5 , 32 , 34 ]. In our earlier study [ 28 , 35 ] thus, we observed overexpression of E-selectin and receptors for IL-8 in dermatitis herpetiformis. TRAIL protein is constitutively present on many cells, but in T and NK cells and dendritic blood cells, it is synthesized after their stimulation [ 13 ].

Some researchers suggest that high level of TRAIL can increase disease activity, whereas some others present opposite opinion. TRAIL can induce the process of apoptosis of dendritic cells and neutrophils and its high expression was seen in T cells infiltrating skin in atopic dermatitis [ 13 ]. The TRAIL expression was significantly higher in lesional than perilesional and control group skin, which may suggest its role in DH pathogenesis.

The influence of TRAIL on human eosinophils did not induce chemotactic effect, but led to apoptosis [ 36 ], therefore, the presence of TRAIL and its receptor in infiltrating cells seems to be related to apoptosis. The recent study of Wu et al. Activation of caspases 3 and 8 critically mediates these processes, but apoptosis can also be triggered. Higher expression of TRAIL receptor in perilesional rather than lesional skin seems interesting, but these results were not statistically significant.

The more intense in uninvolved than in lesional skin expression of TRAIL receptor DR4 on keratinocytes and cells of inflammatory infiltration may suggest its role in maintaining inflammatory process and damage to dermoepidermal junction.

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The role of the process of apoptosis is investigated in the pathogenesis of many autoimmune diseases; however at present, there is not much information about its role in dermatitis herpetiformis. Skin biopsies were taken from 18 DH patients and from 10 healthy subjects. Expression of Bax, Fas, and Fas ligand was detected in the keratinocytes in skin biopsies from DH patients. The expression of examined molecules in biopsies from healthy people was observed only in single cells.

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The Expression of Selected Proapoptotic Molecules in Dermatitis Herpetiformis

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